The Hydrogen Binding Site in Hydrogenase: 35-GHz ENDOR and XAS Studies of the Ni-C Active Form and the Ni-L Photoproduct

Joyce P. Whitehead, Csaba Bagyinka, Michael J. Maroney, Ryszard J. Gurbiel, Brian M. Hoffman*, Ryszard J. Gurbiel, Csaba Bagyinka, Michael J. Maroney

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


EPR, 35-GHz ENDOR, and X-ray absorption (XAS) spectroscopic studies of H2 binding in hydrogenase from Thiocapsa roseopersicina are reported. These studies involve spectra taken on an active form of the enzyme that displays an EPR signal designated Ni-C. This form of the enzyme is light sensitive and is converted to a photoproduct that exhibits a unique EPR signal, Ni-L. 1H-ENDOR spectra reveal two sets of protons that interact strongly with the EPR-active center in Ni-C. The first proton set (H1) is solvent exchangeable, originates from dihydrogen, and has a value of A(H1) ~ 20 MHz. The second proton set (H2) is not solvent exchangeable, has a coupling constant of A(H2) ~ 12 MHz, and is assigned to one or more cysteine β-CH2 protons. Upon irradiation and conversion of the EPR signal to Ni-L, the exchangeable H1 proton set is no longer observed. Upon annealing and regeneration of Ni-C, the feature associated with the H1 proton set is again observed. This is direct evidence that the photoprocess and annealing involve dissociation of a hydrogenic proton and recombination. XAS spectra taken on samples poised to maximize Ni-C and Ni-L do not reveal any significant structural difference between the Ni sites.

Original languageEnglish (US)
Pages (from-to)5629-5635
Number of pages7
JournalJournal of the American Chemical Society
Issue number13
StatePublished - Jun 1 1993

ASJC Scopus subject areas

  • Catalysis
  • Chemistry(all)
  • Biochemistry
  • Colloid and Surface Chemistry


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